title: Plasma membrane calcium ATPase activity is regulated by actin oligomers through direct interaction.
abstract: As recently described by our group, plasma membrane calcium ATPase (PMCA) activity can be regulated by the actin cytoskeleton. In this study, we characterize the interaction of purified G-actin with isolated PMCA and examine the effect of G-actin during the first polymerization steps. As measured by surface plasmon resonance, G-actin directly interacts with PMCA with an apparent 1:1 stoichiometry in the presence of Ca(2+) with an apparent affinity in the micromolar range. As assessed by the photoactivatable probe 1-O-hexadecanoyl-2-O-[9-[[[2-[(125)I]iodo-4-(trifluoromethyl-3H-diazirin-3-yl)benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphocholine, the association of PMCA to actin produced a shift in the distribution of the conformers of the pump toward a calmodulin-activated conformation. G-actin stimulates Ca(2+)-ATPase activity of the enzyme when incubated under polymerizing conditions, displaying a cooperative behavior. The increase in the Ca(2+)-ATPase activity was related to an increase in the apparent affinity for Ca(2+) and an increase in the phosphoenzyme levels at steady state. Although surface plasmon resonance experiments revealed only one binding site for G-actin, results clearly indicate that more than one molecule of G-actin was needed for a regulatory effect on the pump. Polymerization studies showed that the experimental conditions are compatible with the presence of actin in the first stages of assembly. Altogether, these observations suggest that the stimulatory effect is exerted by short oligomers of actin. The functional interaction between actin oligomers and PMCA represents a novel regulatory pathway by which the cortical actin cytoskeleton participates in the regulation of cytosolic Ca(2+) homeostasis.
authors.affiliation: Instituto de Química y Fisicoquímica Biológicas, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, CONICET, Junín 956 (1113) Buenos Aires, Argentina.
authors.forename: Marianela G
authors.initials: MG
authors.lastname: Dalghi
authors.forename: Marisa M
authors.initials: MM
authors.lastname: Fernández
authors.forename: Mariela
authors.initials: M
authors.lastname: Ferreira-Gomes
authors.forename: Irene C
authors.initials: IC
authors.lastname: Mangialavori
authors.forename: Emilio L
authors.initials: EL
authors.lastname: Malchiodi
authors.forename: Emanuel E
authors.initials: EE
authors.lastname: Strehler
authors.forename: Juan Pablo F C
authors.initials: JP
authors.lastname: Rossi
mesh_terms: D000199:Actins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D004910:Erythrocyte Membrane / Q000737:chemistry* / Q000378:metabolism; D006706:Homeostasis / Q000502:physiology*; D006801:Humans; D017136:Ion Transport / Q000502:physiology; D053499:Plasma Membrane Calcium-Transporting ATPases / Q000737:chemistry* / Q000378:metabolism; D055503:Protein Multimerization / Q000502:physiology*; D011817:Rabbits
chemical_list: D000199:Actins; D053499:Plasma Membrane Calcium-Transporting ATPases; D002118:Calcium
keywords: ATPases; Actin; Calcium; Calmodulin; Cytoskeleton; Transport